Abstract

The amniote middle ear is a classical example of the evolutionary novelty. Although paleontological evidence supports the view that mammals and diapsids (modern reptiles and birds) independently acquired the middle ear after divergence from their common ancestor, the developmental bases of these transformations remain unknown. Here we show that lower-to-upper jaw transformation induced by inactivation of the Endothelin1-Dlx5/6 cascade involving Goosecoid results in loss of the tympanic membrane in mouse, but causes duplication of the tympanic membrane in chicken. Detailed anatomical analysis indicates that the relative positions of the primary jaw joint and first pharyngeal pouch led to the coupling of tympanic membrane formation with the lower jaw in mammals, but with the upper jaw in diapsids. We propose that differences in connection and release by various pharyngeal skeletal elements resulted in structural diversity, leading to the acquisition of the tympanic membrane in two distinct manners during amniote evolution.

Comparison of middle ear phenotypes induced by inhibition of the Edn1-Dlx5/6 cascade in mouse and chicken.

(a) Development and morphology of amniote middle ears. Generalized scheme of the first (PA1) and second (PA2) pharyngeal arches (left), transverse section of the middle ear in mammals (middle) and diapsids (right). Three ear ossicles, malleus (m), incus (i) and stapes (st) are present in mammals, while diapsids have only a single ossicle, columella auris (col). The TM comprises the first pharyngeal pouch (PP1) and external auditory meatus (EAM). Left lateral appearance (b,c,f,g), horizontal sections (d,h) and ventrolateral views of the three-dimensional (3D) reconstructed middle ear (e,i) in control (b–e) and Ednra-null (f–i) mice. Higher magnification of boxes in b,f are shown in c,g, respectively. Skeletal components derived from PA1 (green) and PA2 (blue), EAM (pink) and PP1 (grey) are shown in e,i. Left lateral appearance (j,k,n,o), horizontal sections (l,p) and ventrolateral views of the 3D reconstructed middle ear (m,q) in control (j–m) and Edn-blocker-treated (n–q) chickens. (k,o) Higher magnifications of j,n, respectively. Outer edges of EAM is delineated by red dashed lines and ventral auricular feathers are indicated by arrowheads (k,o); they delineate the ventral margin of the EAM in control (k) and the supernumerary EAM was formed ventrally to them (o). Note that pigmentation is also impaired by Edn blockage, which acts on the Ednrb receptor in melanocytes (n,o). White circles and lozenges indicate the pterygoid and otic process of the quadrate, respectively, and TM is coloured translucent yellow (m,q). The extracolumella (a lateral part of the columella auris that attaches to the TM in control) is duplicated and attached to the ectopically formed TM in Edn-blocker-treated chicken embryos (m,q,r; also see ). (r) Summary of the induced middle ear phenotypes in chicken embryos. Dark brown ovals indicate the ventral auricular feathers. art, articular; at, ala temporalis; au, auricle; ec, extracolumella; lj, lower jaw; PJJ, primary jaw joint; q, quadrate; sp, styloid process; ty, tympanic ring; uj, upper jaw; *duplicated elements. Scale bars, 500 μm for c,g, 1 mm for other panels.

(a) In ancestral amniotes, the position of PJJ is thought to have been distant from PP1. The TM had not yet been obtained. (b) In mammalian ancestors, the PJJ shifted dorsally to the proximity of PP1, leading to the coupling of the TM and lower jaw specifications to form the Edn1-Dlx5/6-dependent TM in the lower jaw domain. This TM spans the angular, dependent on Gsc expression. (c) In modern diapsids, the position of the PJJ retains the ancestral state. The hyomandibular has been decoupled from the quadrate and established a connection with the Edn1-Dlx5/6-independent TM in the upper jaw domain. ang, angular; art, articular; ap, ascending process; at, ala temporalis; col, columella auris; hm, hyomandibular; i, incus; m, malleus; Mc, Meckel's cartilage; PP1, first pharyngeal pouch; pq, palatoquadrate; q, quadrate; st, stapes; ty, tympanic ring.